Results of December 2012 - October 2013 Sampling Events ... · Bonnie Mull, MPH Regional...

Results of December 2012 - October 2013 Sampling Events

Orange County, FL Naegleria fowleri Environmental Factors Longitudinal Study

Vincent R. Hill, PhD, PE

Team Lead; Water, Sanitation and Hygiene Laboratory Team

Centers for Disease Control and Prevention

Division of Foodborne, Waterborne, and Environmental Diseases

Waterborne Disease Prevention Branch

1600 Clifton Rd NE

Mailstop D-66

Atlanta, GA 30329

Phone: 404-718-4151

[email protected]

Bonnie Mull, MPH

Regional Environmental Epidemiologist

Food & Waterborne Disease Program

Florida Department of Health

224 SE 24th St

Gainesville, FL 32641

(352) 334-8836 (Office)

(850) 210-5200 (Cell)

[email protected]

Disclaimer

The findings and conclusions in this report are those of the authors and should not be construed

to represent any agency determination or policy. Use of trade names and commercial sources is

for identification only and does not imply endorsement by CDC.

mailto:[email protected]

2

STUDY SUMMARY

Beginning in December 2012, the Florida Department of Health (DOH) - Orange County and

Food and Waterborne Disease Program staff collaborated with scientists in the CDC Waterborne

Disease Prevention Branch (WDPB) to conduct environmental testing of samples from lakes in

the Orlando metro-area over a period of 11 months. The goal of this investigation was to

improve scientific understanding of Naegleria fowleri in natural water bodies and to assess

physical, chemical and biological factors contributing to its presence and concentration over

time. For this study, DOH staff collected 1-L water and sediment samples from 9 lakes in

December 2012, June and October, 2013 in Orange County, Florida (Figure 1). Additionally, two

lakes were sampled more frequently (n=10) over the study period. At the time of sample

collection, the field team measured water temperature, pH, specific conductance, and dissolved

oxygen (DO). Each 1-L sample was a composite of 4, 250-mL samples collected from a bathing

area at each lake. Additional water samples were also collected for bacterial and chemical

testing. All samples were shipped priority overnight for testing at CDC by scientists in the

WDPB Environmental Microbiology Laboratory. The lake samples were tested using the

methods reported in Mull, Jothikumar, and Hill, 2013. In short, water samples were centrifuged

to pellet N. fowleri trophozoites and cysts. After washing the sediment samples in WB saline, the

supernatants were processed using the same procedures as performed for the water samples.

Immunomagnetic separation (IMS) was used to separate N. fowleri trophozoites and cysts from

other amebas and other water constituents. After IMS, each sample was assayed by real-time

polymerase chain reaction (PCR) (to detect and estimate the concentration of N. fowleri) and by

culture (for N. fowleri isolation). The sample pellets were also cultured directly without IMS

processing. Positive N. fowleri detection was confirmed using a second PCR assay (Qvarnstrom

et al, 2006).

The CDC and Florida DOH team is in the process of analyzing these data and plan to report the

results in a peer-reviewed journal manuscript. This report was prepared as a summary of the

laboratory results for consideration by DOH and has not been subjected to statistical analysis or

peer review.

Other water quality parameters tested but data not shown in this report include specific

conductance, dissolved oxygen, total suspended solids, turbidity, total organic carbon, calcium

and magnesium hardness, total nitrogen, total iron, manganese, and total phosphorus.

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Figure 1. Location of Study Lakes in Orange County, FL

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Table 1. Characteristics of lakes sampled for presence of Naegleria fowleri, December 2012-October 2013

Sample site Surface area (acres) Mean Depth (feet) Volume (gallons) Watershed Lake Region

Baldwin 196 15 9.87 x 108 Little Econ Orlando Ridge

Moss 1,135 7 2.55 x 109 Lake Hart Osceola Slope

Downey 18 16 9.38 x 107 Little Econ Eastern Flatland

Jessamine 292 16 1.53 x 109 Boggy Creek Orlando Ridge

Conway 1,773 23 9.48 x 109 Boggy Creek Orlando Ridge

Kelly Unk Unk Unk Wekiva River Unk

Ft Maitland 449 Unk Unk Howell Branch Orlando Ridge

Dinky Dock 225 Unk Unk Howell Branch Orlando Ridge

Keene 1,579 14 7.43 x 109 Cypress Creek Doctor Phillips

Provided by: Orange County Water Atlas at http://www.orange.wateratlas.usf.edu/waterresourcesearch.aspx

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Table 2. N. fowleri presence/absence test results for 9-lake dataset

12/11/12 6/4/13 10/1/13

Sample site Sample type IMS w/o IMS IMS w/o IMS IMS w/o IMS

Direct Culture Culture Direct Culture Culture Direct Culture Culture

Baldwin Water Neg Neg Neg Neg Neg Neg Neg Neg Neg

Sediment Neg Neg Neg Neg Neg Neg Neg Neg Neg

Moss Water Neg Neg Neg Neg Neg Neg Neg Neg Neg

Sediment Neg* Neg* Neg* Neg Neg Neg Neg Neg Neg

Downey Water Neg Neg Neg Neg Neg Neg Positive Neg Neg

Sediment Positive* Neg* Positive* Neg Neg Neg Neg Neg Neg

Jessamine Water Neg Neg Neg Positive Neg Neg Positive Neg Neg

Sediment Neg* Neg* Neg* Neg Neg Positive Neg Neg Neg

Conway Water Neg Neg Neg Neg Neg Neg Neg Neg Neg

Sediment Neg* Neg* Neg* Neg Neg Neg Neg Positive Neg

Kelly Water Neg Neg Neg Neg Neg Neg Neg Neg Neg


Ft Maitland Water Neg Neg Neg Neg Neg Neg Neg Neg Neg


Dinky Dock Water Neg Neg Neg Neg Neg Neg Neg Neg Neg


Keene

Water Neg Neg Neg Neg Neg Neg Neg Neg Neg


*Water was collected from the lake bottom above the sediment; Neg= Negative

On December 11, 2012, one sampling team collected water from the lake bottom above the sediment instead of sediment at Moss, Downey,

Jessamine, and Conway lakes.

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Table 3. N. fowleri direct detection test results

Sample site Sample type 12/11 1/3 2/12 3/5 4/2 4/23 5/7 6/4 6/18 7/2 7/30 8/18 9/10 10/1

Baldwin Water Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Positive Positive Positive Neg

Sediment Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Postive Positive Positive Neg

Downey Water Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Positive Positive Positive

Sediment Positive* Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg


Table 4. N. fowleri culture detection with IMS test results


Baldwin Water Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Positive Neg Neg Neg

Sediment Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

Downey Water Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Positive Neg Neg

Sediment Neg* Neg Neg Neg Neg Neg Neg Neg Neg Neg Positive Positive Neg Neg


Table 5. N. fowleri culture detection without IMS test results


Baldwin Water Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Positive Neg Neg Neg

Sediment Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

Downey Water Neg Neg Neg Positive Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

Sediment Positive* Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Positive Neg Neg

* Water was collected from the lake bottom above the sediment; Neg= Negative

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22

23

24

25

26

27

28

29

30

31

Dec 11 Jun 4 Oct 1

Te

mp

era

ture

(˚C

)

Sample Date

Figure 2. Surface water temperature for the 9-lake dataset

Baldwin

Moss

Downey

Jessamine

Conway

Kelly

Ft.

Maitland

Dinky

Dock

Keene

8

S= Surface; B=Bottom just above sediment

5

5.5

6

6.5

7

7.5

8

8.5

9

Dec 11 Jun 4 Oct 1

pH

val

ue

Sample Date

Figure 3. pH values for the 9-lake dataset

Baldwin S

Baldwin B

Moss S

Moss B

Downey S

Downey B

Jessamine S

Jessamine B

Conway S

Conway B

Kelly S

Kelly B

Ft. Maitland S

Ft. Maitland B

Dinky Dock S

Dinky Dock B

Keene S

Keene B

9


0

20000

40000

60000

80000

100000

120000

140000

Dec 11 Jun 4 Oct 1

CFU

/10

0m

L

Sample Date

Figure 4. Total coliform concentrations for the 9-lake dataset

Baldwin S

Baldwin B

Moss S

Moss B

Downey S

Downey B

Jessamine S

Jessamine B

Conway S

Conway B

Kelly S

Kelly B

Ft. Maitland S

Ft. Maitland B

Dinky Dock S

Dinky Dock B

Keene S

Keene B

10

0

1000

2000

3000

4000

5000

6000

Dec 11 Jun 4 Oct 1

CFU

/100

mL

Sample Date

Figure 5. E. coli concentrations for the 9-lake dataset

Baldwin S

Baldwin B

Moss S

Moss B

Downey S

Downey B

Jessamine S

Jessamine B

Conway S

Conway B

Kelly S

Kelly B

Ft. Maitland S

Ft. Maitland B

Dinky Dock S

Dinky Dock B

Keene S

Keene B


11


0

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

Dec 11 Jun 4 Oct 1

CFU

/mL

Sample Date

Figure 6. Heterotrophic plate count concentrations for the 9-lake dataset

Baldwin S

Baldwin B

Moss S

Moss B

Downey S

Downey B

Jessamine S

Jessamine B

Conway S

Conway B

Kelly S

Kelly B

Ft. Maitland S

Ft. Maitland B

Dinky Dock S

Dinky Dock B

Keene S

Keene B

12

14

16

18

20

22

24

26

28

30

32

34

36

Dec 11 Jan 3 Feb 12 Mar 5 Apr 2 Apr 23 May 7 May 21 Jun 4 Jun 18 Jul 2 Jul 30 Aug 18 Sep 10 Oct 1

Tem

per

atu

re (˚

C)

Sample Date

Figure 7. Water temperatures for 2-lake dateset

Baldwin Surface

Baldwin Bottom

Downey Surface

Downey Bottom

13

0

100,000

200,000

300,000

400,000

500,000

600,000

700,000

800,000


CFU

/100

mL

Sample Date

Figure 8. Total coliform concentrations for 2-lake dataset

Baldwin Surface

Baldwin Bottom

Downey Surface

Downey Bottom

14

0

20,000

40,000

60,000

80,000

100,000

120,000


CFU

/10

0m

L

Sample Date

Figure 9. E. coli concentrations for 2-lake dataset

Baldwin Surface

Baldwin Bottom

Downey Surface

Downey Bottom

15

0

20,000

40,000

60,000

80,000

100,000

120,000

140,000

160,000

180,000

200,000

220,000


CFU

/mL

Sample Date

Figure 10. Heterotrophic plate count concentrations for 2-lake dataset

Baldwin Surface

Baldwin Bottom

Downey Surface

Downey Bottom

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DISCUSSION

The December 2012 to October 2013 lake sampling data from Orange County have helped CDC

evaluate the effectiveness of new sample processing and analytical methods for detecting N.

fowleri in water and sediment samples. The environmental N. fowleri detection data summarized

in this report will be useful to CDC in conjunction with future ecological studies of N. fowleri.

The sampling methods used in this study and associated analytical results should be useful to

Florida DOH public health officials for future Naegleria investigations.

Seventeen of the 106 samples (16%) were positive for N. fowleri by at least one test method. The

frequency of positive detection was similar for both sample types; eight were sediment samples

and nine were water samples. Downey Lake had the highest frequency (23%) of positive samples

followed closely by Baldwin Lake (20%). Twelve of the samples were positive for N. fowleri

when the PCR test was conducted directly on the sample without culture. These positive direct

detection test results indicate the presence of N. fowleri DNA in the sample, however it cannot

differentiate between viable or dead N. fowleri cells. Three of those twelve samples were also

positive for culturable thermophilic amebas, confirmed to be N. fowleri by PCR. This could be

due to overgrowth of more rapidly growing but nonpathogenic free-living ameba species or that

the N. fowleri cells were no longer viable. Five additional samples were determined to be

positive for N. fowleri after culture that were negative by direct detection testing. It is not clear

whether this is due to the sample volume tested (which was lower for PCR than for culture), PCR

inhibition, or other factors. N. fowleri was detected when the average water temperature was 29.9

° C (±4.1° C) compared to the average water temperature of 25.9° C (±4.1° C) when N. fowleri

was not detected. On average, total coliform concentrations were slightly higher in N. fowleri

positive samples (8.8 x 104 CFU/100mL) compared to samples in which N. fowleri was not

detected (2.5 x 104 CFU/100mL), but these data have not been statistically analyzed. The data for

this project suggest that water temperatures and bacterial concentrations may be factors

contributing to detection of N. fowleri in some lakes in Orange County. It is still not clear why

N. fowleri was detected in some Orange County lakes, but not others. It is also difficult to

interpret the negative test results since the results are from relatively few samples; limited data

on Naegleria sampling methods exists to understand how sample test results are affected by lake

location, time of day, season, or location in the water column. Test results may have been

different under other conditions or if more intensive sampling had occurred.

When considering the data in this report, it is important to note that Naegleria fowleri is

normally found in the natural environment and is well adapted to surviving in various habitats,

particularly warm-water environments. There is no established relationship between detection or

concentration of Naegleria fowleri and risk of infection.Therefore the data reported in this

document are not useful for public health risk estimation. Environmental investigations, such as

the investigation in Florida discussed in the present report, are useful for building an evidence

base that may assist scientists to better understand the environmental dynamics of N. fowleri,

factors influencing the ameba’s presence and growth, and its geographical distributions in new

environments.

Additional research such as the present study are needed to develop a more extensive evidence

base that can help scientists better understand the relationship between environmental factors and

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the presence and concentration of N. fowleri in lake environments. Cases of primary amebic

meningoencephalitis (PAM) are known to correlate with warm weather (and, by association,

warm water temperatures). However, within regions and states, it is not known why cases of

PAM are associated with certain water bodies but not others that also have Naegleria present.

We hope to continue to conduct ecological studies to investigate various environmental factors

that may be associated with the distribution of PAM case exposures in the United States. Work is

planned to perform statistical analysis of the water, sediment and climatological data collected

for this study, with the intent to publish findings from the analysis. CDC will engage with

Florida DOH public health officials on the analyses and plans to report the results in a peer-

reviewed journal manuscript.

REFERENCES

Mull BJ, Jothikumar N and Hill VR. (2013) Improved method for the detection and

quantification of Naegleria fowleri in water and sediment using immunomagnetic

separation and real-time PCR. J Parasitol Res, Article ID 608367, doi:

10.1155/2013/608367.

Qvarnstrom Y, Visvesvara GS, Sriram R, and da Dilva AJ. (2006) Multiplex real-time PCR

assay for simultaneous detection of Acanthamoeba spp., Balamuthia mandrillaris, and

Naegleria fowleri. J Clin Microbiol, 44(10):3589-3595.

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